The ability of the immune system to generate immense repertoires of clonally distributed antigen receptors depends on the process of V(D)J recombination, which occurs in a highly regulated fashion during the early stages of T and B lymphocyte development. A critical aspect of developmental regulation is the process of allelic exclusion, which limits certain antigen receptor loci to produce a functionally rearranged gene on only one allele. Allelic exclusion is enforced in part through feedback inhibition in which a productive VDJ recombination event on one allele blocks further V to DJ recombination on the second allele. The mechanisms responsible are not well understood. We propose that feedback inhibition associated with TCR beta allelic exclusion is multifactorial, with several influences converging to suppress V beta to DJ beta recombination. Feedback was thought to be enforced by downregulation of V beta accessibility. However, we found that feedback is intact even after the accessibility barrier has been overcome. We propose that this "beyond accessibility" regulation may reflect changes in locus conformation that inhibit recombination events between distal recombination signal sequences (RSSs), changes in locus subnuclear position that may restrict the availability of the recombinase, and unique properties of RSSs or factors recruited to their vicinity that may provide developmental specificity to RSS usage. In Specific Aim I we will identify developmental changes in TCR beta locus subnuclear position and conformation using the approach of three dimensional fluorescence in situ hybridization. In Specific Aims II and III we will use complementary gene- targeting approaches to directly test mechanisms of feedback inhibition. First, we will create and study compact V(D)J recombination cassettes embedded in the TCR beta and TCR alpha loci that should override any contributions of locus conformation and subnuclear position. Second, we will modify local elements within the TCR beta locus that should override potential contributions from local regulation. Finally, in Specific Aim IV we will define the mechanisms that segregate the TCR beta locus into distinct chromatin regulatory units. Successful completion of these studies should provide general lessons regarding the control of V(D)J recombination and the mechanisms that guide the formation of a clonally distributed and non-autoreactive antigen receptor repertoire.